Development and Validation of a Simple, Rapid and Inexpensive PCR-RFLP Method for Genotyping of Common IL28B Polymorphisms: A Useful Pharmacogenetic Tool for Prediction of Hepatitis C Treatment Response

Abstract

Implication for health policy/practice/research/medical education:This article focuses on development of a simple method for genotyping of IL28B polymorphisms. This study is recommended to researchers in the field of viral hepatitis. Background:In 2009, 3 genome-wide association studies implicated IL28B single-nucleotide polymorphisms (SNPs) as the strongest genetic pretreatment predictor of sustained virological response (SVR) in hepatitis C infection. Recently, the American Association for the Study of Liver Diseases (AASLD) and the European Association for the Study of the Liver (EASL) included IL28B testing in their guidelines. Objectives: The main aim of this study was to develop and validate a simple, rapid, and inexpensive polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method for genotyping of common IL28B polymorphisms (rs12979860 and rs8099917). Patients and Methods: Two methods were developed to genotype common IL28B polymorphisms: 1) PCR-sequencing as a reference method and 2) PCR-RFLP as a rapid and inexpensive method. Both polymorphisms were genotyped in 104 Iranian hepatitis C patients by both methods simultaneously. To validate the PCR-RFLP method, the PCR-RFLP genotyping results should be 100% concordant with the PCR-sequencing results. Results: Genotyping of rs12979860 and rs8099917 by PCR-RFLP was concordant with PCR-sequencing in 104 (100%) individuals. The analytical sensitivity and specificity of the PCR-RFLP method for genotyping of both SNPs are 100%. Among these 104 patients with chronic hepatitis C, the frequency of the rs12979860 CC, CT and TT genotypes were 40.4%, 47.1% and 12.5% and the frequency of the rs8099917 TT, GT and GG genotypes were 59.6%, 35.6% and 4.8%, respectively. Also, three IL28B haplotypes (rs12979860-rs8099917) were found among our patients including C-T, T-G and T-T with 63.9%, 22.6% and 13.5% frequency, respectively. C-G haplotype was absent in all of our patients. Conclusions: We have developed a validated, fast, and simple PCR-RFLP method for genotyping of common IL28B SNPs that is more cost-effective than sequencing.

Letter: the rs12979860 and ss469415590 polymorphisms of IFNL4 gene are in strong linkage disequilibrium in Caucasian patients with chronic hepatitis C

“…1 We also assessed the rs12979860 and ss469415590 SNPs in 183 Iranian patients with hepatitis C by DNA sequencing. 2 In our study population, we found the frequency of ss469415590 TT/TT, TT/DG and DG/DG to be 38.8%, 45.4% and 15.8%, respectively, and the distribution of rs12979860 C/C, C/T and T/T genotypes to be 38.8%, 45.4% and 15.8%, respectively, which resulted in strong linkage disequilibrium (r 2 = 1.0) between the ss469415590 and rs12979860 SNPs. The perfect correlation of these two genetic variants in Caucasian Iranian patients was similar to that in the study by Stattermayer et al…”

Genetic Variation in Interleukin-28B and Response to Peg-IFNα-2a/RBV Combination Therapy in Patients with Hepatitis C Virus Infection

“…It is noteworthy that PCR-RFLP assay has been utilized to determine this polymorphism in several studies such as those conducted by Sharafi et al (23), Irman et al (24), Fateh et al (16), and Nakamoto et al (25). These studies have reported that this assay is simple, fast, inexpensive, and valid.…”

High Response Rate to Pegylated Interferon Alpha and Ribavirin Combination Therapy in Hemophilic Children with Chronic Hepatitis C; A Case-Control Study

“…In this study, rs12979860 polymorphism was assessed as the most common polymorphism near IL28B (IFNL3) gene. The detailed protocol of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method for genotyping of rs12979860 polymorphism was previously described [8]. The patients did not undergo liver biopsy because determining of histological severity is not obligatory before HCV treatment and also this procedure is life threatening and costly in patients with congenital bleeding disorders.…”